Reactor module with capillary membranes

ABSTRACT

The present invention relates to a reactor module constructed of hollow fibers and cells as well as to reactors comprising this reactor module.

This application is based on PCT/EP01/04271, filed Apr. 14, 2001, andclaims priority to German Patent Application Number 10023505.0, filedMay 13, 2000.

FIELD OF THE INVENTION

The present invention describes a reactor module able to function as acomponent of an artificial organ as well as a reactor containing thisreactor module.

BACKGROUND OF THE INVENTION

Diseases, especially of internal organs, occur often in humans and areoften life-threatening. Organ transplantations are often needed, andoften fail due to the limited availability of natural replacementorgans. External systems for supporting the damaged or failed organfunctions often prove to be insufficient, since they significantlyreduce the patient's quality of life, and are also unable to participatein the biochemical processes of the body. Looking at the example of theliver, during recent years, hybrid life support systems have beendeveloped. With these systems, liver cells are cultivated in artificialmodules, whereby different metabolism, elimination, and synthesisprocesses of the liver may be replaced in patients with acute liverfailure. The existing systems are based on an extracorporal circuit towhich the patient is connected. The reactor used consists of a housingin which the liver cells are located. The patient's blood or plasma isin direct contact with the cells. It is hereby known that thehepatocytes are immobilized in or on small spheres, for example alginate(Selden et al., Ann N Y Acad Sci (1999) 875, 353-363; Naka et al,Artificial Organs (1999) 23, 822-828, and Sakai et al., CellTransplantation (1999) 8, 531-541). Also known is the simulation of theparenchymal structure of the liver with multi-dimensionally arrangedcapillary bundles (Custer and Mullon, Adv Exp Med Biol (1998) 454,261-271; Busse and Gerlach, Ann N Y Acad Sci (1999) 875, 326-339,Flendrig et al., Int J Artif Organs (1999) 22, 701-708; Margulis et al.,Resuscitation (1989) 18, 85-94; Ellis et al., Hepatology (1996) 24,1446-1451; Gerlach et al., Transplantation (1994) 58, 984-988).

The described artificial organs, in which natural cells are immobilizedon a sub-structure, for example polymer hollow fibers or capillaries,have the disadvantage that the cells often can only be immobilized withdifficulties. The reason for this is, among other things, that thefluctuations in diameter that are associated with flow-through pulses inthe polymer hollow fibers usually make immobilization more difficult andalso could lead to denaturation effects in the cells.

SUMMARY OF INVENTION

The present invention is therefore based on the technical problem ofmaking available a reactor module for use in artificial organs thatovercomes said disadvantages.

The present invention solves the underlying technical problem byproviding a reactor module, comprising at least one ceramic hollow fiberand at lest one biological cell, whereby at least one biological cell isimmobilized on the surface of the at least one ceramic hollow fiber. Inan especially preferred embodiment, the at least one biological cell isa liver cell, i.e. a hepatocyte. Naturally, other cells, for examplerenal cells, conjunctive tissue cells, fibroblasts, immune cells,intestinal cells, skin cells, pancreatic cells, spleen cells, or bloodcells also can be used according to the invention.

In another preferred embodiment of the invention, many cells, inparticular a cell layer, in particular a monolayer, are immobilized onthe surface of the at least one ceramic hollow fiber.

The invention is among other reasons advantageous in that the inherentstiffness of the used ceramic hollow fibers enables its defined spatialpositioning within a reactor space. In contrast to polymer hollow fibersor capillaries, this does not result in fluctuations in the diameterassociated with flow-through pulses, so that the immobilized cellscannot be negatively affected. The ceramic hollow fibers can be adaptedwith respect to their geometry, their outer and inner diameter, andtheir porosity and pore size to any cell species, thus making thereactor module according to the invention suitable for manyapplications. In addition, the ceramic hollow fiber provides a surfacethat can be modified with many different processes of a physical andelectrical or chemical nature. This makes it possible to achieveimproved immobilization of the cells. Finally, ceramic hollow fibershave pores that enable a removal of metabolic products and, as the casemay be, also the supply of nutrients.

According to the invention, it is provided that the cells to beimmobilized are brought into contact with the ceramic hollow fibers,grow onto the surface of the hollow fibers, proliferate, and form amonolayer. The toxic metabolic products emitted by the immobilized cellsare able to reach the inside of the hollow fibers via the defined porescontained in the hollow fibers, and can be removed from there, or can besupplied via the defined pores with nutrients from inside the hollowfibers.

In connection with the present invention, a biological cell means thestructural and functional unit of the organisms, which is characterizedby its growth, proliferation, and metabolic capability. Such cells canbe eukaryotic cells, such as animal, plant, or yeast cells. Inconnection with the present invention, cells also mean prokaryoticcells, such as bacteria cells. In an especially advantageous embodiment,the cells are human or animal cells, in particular liver cells,fibroblasts, connective tissue cells, intestinal cells, blood cells,immune cells, skin cells, spleen cells, kidney cells, or pancreaticcells. Naturally, the cells may also be naturally occurring cells orcells manipulated with gene technology. The cells may be healthy ordiseased, for example immortalized or carcinogenic. The cells may bedifferentiated or dedifferentiated, omnipotent, pluripotent, orunipotent.

In connection with the present invention, an immobilization means aspatial fixation of the at least one biological cell on or at thesurface of the ceramic hollow fiber. The immobilization may bereversible or irreversible. It may be performed by simple cultivationand growing to the surface of the hollow fiber, but may also be broughtabout or accelerated with chemical or physical processes.

In connection with the present invention, a ceramic hollow fiber means ahollow fiber made from ceramic materials, i.e. a hollow fiber consistingof inorganic and primarily non-metallic compounds or elements thatpreferably contains more than 30% by volume of crystalline materials.According to the invention, both oxidic as well as non-oxidic ceramicmaterials can be used. Such non-oxidic materials, for example, mayinclude silicium carbide SiC or silicium nitride Si₃N₄ that may beproduced, for example, by pyrolysis from polycarbosilanes or,respectively, from polysilazanes. Naturally, it is also possible toproduce oxidic ceramic membranes with hollow fiber geometry, whereby,for example, a ceramic powder is mixed with a binder, and this pastymass is extruded. This is followed by sintering, whereby a dense greenfiber with a ceramic structure is produced, whose pore size may bevaried depending on various parameters, such as amount and nature of thesintered binder, the sinter regime, the powder morphology, etc.

It may also be provided that ceramic materials with cellulose as abinder are used, whereby these are dissolved in a solvent, for exampleN-methyl-morpholine-N-oxide (NMMNO). This cellulose solution is mixedwith ceramic powder and further processed in an actually known manner(DE 4426966 TITK of Feb. 1, 1996).

In another embodiment, the invention furthermore relates to a previouslymentioned reactor module, whereby the capillary membranes have an innerdiameter of 0.1 to 4 mm, preferably 0.5 to 1 mm. In another embodiment,the pore size of the hollow fiber used in the reactor module accordingto the invention is 0.05 to 1 μm, preferably 0.1 to 0.5 μm.

In another embodiment of the present invention, the surface of thehollow fibers is modified. The modification provided according to theinvention permits an improved immobilization of the cells in vitro. Thesurface modification may be performed using chemical and physical, inparticular thermal or electrical processes. It may be provided, forexample, to use chemical processes such as radical or ionic graftcopolymerization, simple coatings, or gas phase processes, such aslow-pressure or low-temperature plasma technology. The use oflow-pressure plasma processes is hereby especially preferred, since itallows both defined changes of the physical surface properties, such asroughness, as well as of the chemical composition of the surface in asingle step. The plasma treatment that is especially preferred accordingto the invention permits a free selection of the substrate material, adefined adjustment of surface properties without changing volumecharacteristics; uses only small amounts of chemicals as a result of thephysical vacuum process, and makes it possible to perform the process ina dry, closed system. Plasma, also an ionized gas with the exact samenumber of positive and negative charges, can be used for ionization overa very wide range of pressures and temperatures. According to anespecially preferred embodiment of the present invention, low-pressureplasma with a pressure from 0.01 to 1 mbar is used for surfacemodification. The plasma atmosphere consists of free electrons,radicals, ions, UV radiation, and a large number of particles excited indifferent manners. By choosing the concentration of the chemicalcomposition of the monomers introduced into the gas chamber, thedwelling time of the molecules in the reaction space, the inputhigh-frequency power, and the electrostatic charge of the treatedmaterial, the formation of the new surface can be determined in adefined manner (Strobel et al., Plasma surface modification of polymers:Relevance to adhesion, Utrecht (1994)). The treatment time providedaccording to the invention may be varied according to requirements;however, in an especially preferred embodiment it ranges from 1 secondto 10 minutes, preferably 4 seconds to 5 minutes. By using thelow-pressure plasma technology provided according to the invention, thesurface can be cleaned and an etching abrasion can be achieved, themicro-roughness can be modified, the formation of radical points andsubsequent secondary reactions, such as cross-linking processes or graftcopolymerization, as well as plasma polymerization and thus theformation of homogeneous, adhesive films in the nanometer range can beachieved.

According to the invention, a preferred embodiment also provides thesurface with functional groups, for example hydrophobic, anionic,cationic, for example amino groups, or polarized groups and/or withpolypeptides or proteins. This includes in particular matrix proteins,such as fibronectin, laminin, or collagen.

According to the invention, the surface of the hollow fibers may beprovided with adhesion promoters, such as integrins, fibronectin,vitronectin, and/or collagen in order to facilitate the immobilizationof the cells.

In an especially preferred embodiment of the present invention, thesurface may be provided with a primer layer with, for example, SiOxstoichiometry. This represents an interface layer—from organic toinorganic, from silicone to glass—to which other groups can be bondedvery easily. This primer coat then can be functionalized withnitrogen-containing groups, for example with functional groups, inparticular amino groups. It would also be conceivable according to theinvention to provide other surfaces, in particular cationic surfaces.Naturally, the primer layer can be connected via spacers or linkers withthe functional groups.

In another embodiment of the present invention, the ceramic hollowfibers are arranged parallel to each other in one plan in a frame,whereby the hollow fibers are connected with each other in series or arepresent separately from each other. The frame may consist of plastic,for example polystyrene, polymethyl methacrylate, polycarbonate, orpolypropylene or may contain substantial parts of it. In a specialembodiment of the present invention, the reactor module according to theinvention is realized in the form of a preferably square or rectangulardisk. The arrangement of the hollow fibers according to the invention inone plane, for example in mats, makes it possible to avoid the risk of“tunneling” associated with the flow through the external space and thusa reduction in efficiency.

In another embodiment, thermal compensation elements, for example offiberglass or carbon fiber strands, may be provided in the frame of thereactor module, said compensation elements accounting both for thermaltensions occurring, for example, during sterilization or freezing, andfor the different materials used.

In another preferred embodiment of the present invention, this alsorelates to a reactor comprising at least one previously mentionedreactor module, particularly in disk shape, and a housing. According tothe invention, several of the preferably disk-shaped reactor modulesmust be arranged in layers at defined intervals in such a way relativeto each other that a required surface can be composed by layering asufficient number of such disk-shaped reactor modules. The inlet andoutlet of the flowing media can be implemented with a connection system,as is used, for example, in standard electrodialysis modules. The endpieces contain the corresponding connections through which theindividual channels, formed by recesses in the disk-shaped reactormodules, can be supplied.

In a preferred embodiment of the present invention, the frames of themodules have openings that may serve as inlet or outlet openings for themetabolic products or nutrients. According to the previously describedembodiment, in which several reactor modules are arranged serially in areactor, the frames of the modules at the same time form the outsidewall of the reactor. The housing is provided in its terminal areas withinlet and outlet openings for the fluid, for example plasma, passingthrough the reactor. Depending on the orientation of the seriallyarranged frames in relation to each other, the inlet and outlet openingsfor the fluid in the terminal areas of the frame present channels in theouter reactor wall that is formed by the frames, through which channelsa targeted addition or removal of substances into certain areas of thereactor is made possible. This means that by placing several reactormodules behind each other in a suitable manner, supply and removalchannels for the reactor inside space, which comprises both theextracapillary and intracapillary space, can be formed, in this wayenabling a functional and spatial compartmentalization. In this way,different, serially provided cultivation conditions may be provided forthe same or different cells in order to enable an organ function that isas efficient and as versatile in its control as possible.

In an especially preferred embodiment of the present invention, thereactor furthermore has oxygenation, heat exchanger and/or samplecollection units or inoculation access points. In an especiallypreferred embodiment of the present invention, the reactor inside spaceis closed off with sterile filters located in the area of the terminalareas in order to prevent a flushing out of cells and cell componentsinto the patient circuit.

The reactor according to the invention may be used, for example, toconstruct an artificial liver, whereby the reactor is connected togetherwith pumps and plasma separators into an extracorporal circuit involvingalso the patient. In such a preferred embodiment, the reactor accordingto the invention is preceded by a plasma separator or is integrated insuch a plasma separator in order to enable a cell-free operation of thereactor.

Other advantageous embodiments of the invention derive from thesecondary claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is explained in detail in reference to the followingexample and associated figures.

FIG. 1 shows a disk-shaped reactor module,

FIG. 2 shows a preferred arrangement of different reactor modulespositioned behind each other,

FIG. 3 is a schematic longitudinal section of the construction of areactor, and

FIG. 4 is a schematic of the construction of an artificial organ.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows a reactor module 1, comprising a plurality of hollow fibersarranged in one plane and parallel to each other, which are clamped intoa square, disk-shaped frame 5. The frame 5 thus encloses the hollowfibers 3 that are arranged in a single plane on all sides and keeps themspatially fixated. At the same time, the frame two-dimensionallyencloses an inside space that contains the ceramic hollow fibers 3, andis divided by said ceramic hollow fibers into one space inside and onespace outside of the hollow fibers 3. The frame 5 has integratedopenings 7 for the addition and removal of fluids and gases (not shown).Both the space inside the hollow fibers 3 and outside the hollow fibersis in fluid connection with the inlet or outlet openings 7, making itpossible to pass fluids, such as gases or preferably liquids, into orout of the capillary inside or outside space in a targeted manner. Theframe 5 furthermore includes thermal compensation elements 9 in the formof two recesses that extend parallel to each other over the entire widthof the reactor module 1, and in each of which recesses a compensationstrip is arranged.

The hollow fibers 3 were produced and surface-modified as follows:N-methyl-morpholine-N-oxide was prepared as a 50% solution in water,into which solution cellulose was added; the cellulose was dispersed,and then part of the water was vacuum-distilled. The resultingsuspension is homogenized, and ceramic powder suspended inN-methyl-morpholine-N-oxide is added. Then the residual water isdistilled off, and the entire suspension is again homogenized andde-gassed, resulting in a homogeneous spinning mass. In a subsequentspinning process, soaked fibers with a stable, hollow structure areobtained. During the spinning process, the homogeneous spinning mass istransferred into a spinning bath, resulting in a phase inversion of thecellulose that is accompanied by a stabilization of the hollowstructure. During the soaking process, water is exchanged forN-methyl-morpholine-N-oxide. The soaked fiber with stabilized hollowstructure obtained during the spinning process is dried, which yields adried base fiber that is sintered, resulting in the hollow fiber 3.

The hollow fibers 3 were coated with a biomatrix—preferably collagen.Then a cell suspension of hepatocytes is applied to the cell carrierframes. By lightly moving the frames, the cells come into contact withthe hollow fibers and adhere to the hollow fibers. Then a second layerof collagen can be applied over the cells. The frames, after having beenapplied with the hepatocytes in this manner, can be cryopreserved inliquid nitrogen for long-term storage.

FIG. 2 is a schematic of a potential arrangement of the reactor modules1. The reactor modules 1 are arranged in parallel planes behind eachother, allowing an efficient flow of the body fluid (not shown) of thepatient through them in arrow direction. Also shown are sterilefiltration modules 11 upstream and downstream from the reactor modules 1according to the invention, which prevent cells and cell components frombeing flushed in and out.

FIG. 3 shows a reactor 13 according to the invention. The reactor 13comprises disk-shaped reactor modules 1 arranged behind each other inparallel planes, whereby the frame 5 of the reactor module 1 at the sametime forms the reactor outside wall, the inlet and outlet openings formchannels (not shown) in the reactor outside wall, and the ceramic hollowfibers are arranged in the reactor inside space 15. Depending on theconstruction of the inlet and outlet openings 7 in the used reactormodules 1 and their arrangement in the reactor 13, compartments that aresubstantially separated in a targeted manner from each other andarranged behind each other can be created, said compartments beingcharacterized by a different cell occupation and/or cultivationconditions. The patient's plasma flows through an inlet opening 19 in areactor end part, i.e. a terminal area 17, into the reactor 13, passesthrough a sterile filter 11 and then through the ceramic hollow fibersof the reactor modules 1 in order to then flow out via another sterilefiltration module 11 through a reactor end part 21 in the opposite partof the reactor and the outlet opening 23 in this reactor end part.

FIG. 4 shows an artificial liver 23, comprising pumps 25, a plasmaseparator 27, a reactor 13 according to the invention, and a heatexchanger 29. The blood of a patient (not shown) is brought intocirculation via pumps 25 and is passed through a plasma separator 27, inwhich blood cells are separated. The resulting plasma flows through thereactor 13 according to the invention, in which the hepatocytes thathave been immobilized on the ceramic hollow fibers perform theirmetabolic function. The plasma then flows back into the patient via heatexchanger 29. Like the plasma separator 27, the heat exchanger 29 mayalso be located inside the reactor 13.

What is claimed as new and desired to be protected by Letters Patent ofthe United States is:
 1. A reactor module, comprising at least oneceramic hollow fiber and at least one biological cell, whereby the atleast one ceramic hollow fiber is clamped into a frame of plastic,whereby thermal compensation elements are integrated in the frame of thereactor module, and whereby the at least one biological cell isimmobilized on the at least one ceramic hollow fiber.
 2. A reactormodule according to claim 1, whereby the biological cell is a livercell.
 3. A reactor module according to claim 2, whereby the surface ofthe hollow fiber is modified.
 4. A reactor module according to claim 3,whereby the surface of the hollow fiber is chemically or physicallymodified.
 5. A reactor module according to claim 4, whereby the surfaceof the hollow fiber is modified by using a low-pressure plasma process.6. A reactor module according to claim 5, whereby the surface of thehollow fiber includes adhesion promoters.
 7. A reactor module accordingto claim 1, whereby the reactor module is realized in the shape of adisk.
 8. A reactor module according to claim 1, whereby the hollow fiberhas an inner diameter of 0.1 to 4 mm.
 9. A reactor module according toclaim 8, whereby the pore size of the hollow fiber is 0.05 to 1 μm. 10.A reactor module according to claim 9, whereby inlet and/or outletopenings are integrated into the frame of the reactor module.
 11. Areactor comprising at least one reactor module according to claim 1 in ahousing.
 12. A reactor according to claim 11, comprising at least oneoxygenator, at least one heat exchanger, and/or at least one samplecollection or loading unit.
 13. A reactor according to claim 12, wherebythe reactor modules are arranged in compartments defined by thearrangement of the inlet and outlet openings.
 14. A reactor according toclaim 13, whereby different cells are present in the compartments.
 15. Areactor according to claim 14, whereby different cultivation conditionsexist in the compartments.